Biochemical and functional characterization of renal cortical brush border and basolateral membranes in dogs

The purpose of the present study is to analyze membrane fluidity, enzyme, phospholipid and fatty acid composition and cholesterol content in the brush border (BBM) and basolateral (BLM) membranes obtained from the renal cortex of normal dogs. All measurements were carried out in samples from the same kidney in order to correlate membrane fluidity with membrane composition. BBM and BLM were obtained separately by MgCl precipitation and gradient centrifugation. The order parameter of membrane fluidity was measured by 1,6-dimethyl-1,3,5-hexatriene (DPH) and 1-trimethylammoniophenyl-DPH. (TMA-DPH) steady-state polarization fluorescence. Total lipids, phospholipids and phospholipid classes, cholesterol content, and fatty acid classes were also measured. Data from BLM enzymatic activity revealed an 11-fold enrichment in Na,K-ATPase, whereas the enrichment factors for the other enzymatic markers were well below the unit, demonstrating the high purity of the preparation obtained. Similarly, BBM showed a 9 times increase in alkaline phosphatase and gamma-glutamyltranspeptidase enrichment, and values of enrichment factors for the other enzymatic markers of about 1. BBM exhibited a higher value of steady-state fluorescence anisotropy and thus a lower fluidity than BLM using either of the fluorescent probes DPH or TMA-DPH. This lower fluidity in both the central hydrophobic zone, and the fluorescent probes DPH or TMA-DPH. This lower fluidity in both the central hydrophobic zone, and the external, more hydrophilic leaflet of BBM in comparison with BLM was consistent with the findings of: (a) a higher cholesterol/protein ratio; (b) a lower phospholipid protein ratio; (c) a higher sphingomyelin/choline glycerophospholipid ratio, and (d) a lower unsaturation degree of the fatty acids.